Bottom Line:
The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit.In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed.The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity.

ABSTRACTTranscription factors are involved in a number of important cellular processes. The transcription factor NF-κB has been linked with a number of cancers, autoimmune and inflammatory diseases. As a result, monitoring transcription factors potentially represents a means for the early detection and prevention of diseases. Most methods for transcription factor detection tend to be tedious and laborious and involve complicated sample preparation, and are not practical for routine detection. We describe herein the first label-free luminescence switch-on detection method for transcription factor activity using Exonuclease III and a luminescent ruthenium complex, [Ru(phen)(2)(dppz)](2+). As a proof of concept for this novel assay, we have designed a double-stranded DNA sequence bearing two NF-κB binding sites. The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit. In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed. The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity. This will allow the rapid and low cost identification and development of novel scaffolds for the treatment of diseases caused by the deregulation of transcription factor activity.

Figure 7: The fold change luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of the digestion mixture containing double-stranded non-NF-κB-binding substrate (0.02 µM) and 40 U of ExoIII as a function of the concentration of the p50 subunit (0, 0.10, 0.20 and 0.40 µM).

Mentions:
The emission spectrum in Figure 6 shows that incubation of the oligonucleotide substrate with BSA did not produce the same emission enhancement (fold change of 1.1) as observed for the p50 subunit. To further provide evidence that the inhibition of ExoIII digestion was due to the selective binding of the p50 subunit, we replaced the oligonucleotide substrate with a DNA sequence that cannot bind to the p50 subunit. The non-NF-κB-binding substrate was incubated in the presence of the p50 subunit and the luminescence spectrum of the ruthenium complex in the presence of the digestion mixture was measured (Figure 7). In the presence of 0.4 µM p50 subunit, a fold change of 2.4 was observed, which was significantly lower than the 8-fold enhancement observed with the wild-type sequence.Figure 7.

Figure 7: The fold change luminescence response of [Ru] (1 µM) in TF buffer solution containing K3[Fe(CN)6] (600 µM) in the presence of the digestion mixture containing double-stranded non-NF-κB-binding substrate (0.02 µM) and 40 U of ExoIII as a function of the concentration of the p50 subunit (0, 0.10, 0.20 and 0.40 µM).

Mentions:
The emission spectrum in Figure 6 shows that incubation of the oligonucleotide substrate with BSA did not produce the same emission enhancement (fold change of 1.1) as observed for the p50 subunit. To further provide evidence that the inhibition of ExoIII digestion was due to the selective binding of the p50 subunit, we replaced the oligonucleotide substrate with a DNA sequence that cannot bind to the p50 subunit. The non-NF-κB-binding substrate was incubated in the presence of the p50 subunit and the luminescence spectrum of the ruthenium complex in the presence of the digestion mixture was measured (Figure 7). In the presence of 0.4 µM p50 subunit, a fold change of 2.4 was observed, which was significantly lower than the 8-fold enhancement observed with the wild-type sequence.Figure 7.

Bottom Line:
The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit.In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed.The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity.

ABSTRACTTranscription factors are involved in a number of important cellular processes. The transcription factor NF-κB has been linked with a number of cancers, autoimmune and inflammatory diseases. As a result, monitoring transcription factors potentially represents a means for the early detection and prevention of diseases. Most methods for transcription factor detection tend to be tedious and laborious and involve complicated sample preparation, and are not practical for routine detection. We describe herein the first label-free luminescence switch-on detection method for transcription factor activity using Exonuclease III and a luminescent ruthenium complex, [Ru(phen)(2)(dppz)](2+). As a proof of concept for this novel assay, we have designed a double-stranded DNA sequence bearing two NF-κB binding sites. The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit. In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed. The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity. This will allow the rapid and low cost identification and development of novel scaffolds for the treatment of diseases caused by the deregulation of transcription factor activity.